This project concerns two transport proteins of the human red cell membrane, band 3, which is responsible for anion and water transport and Na,K-ATPase which couples the energy source to cation transport. We plan to investigate: coupling between these two proteins, coupling between them and the lipid matrix and the relationship of water transport to band 3. a) Protein-protein interactions. We have previously shown by 31P NMR that there is a amplification factor of 10 to the 5th power by which responses induced in the 200 copies/cell of the Na,K-ATPase are reflected in changes in a large fraction of the 500,000 dimers/cell of band. 3. We will use fluorescent probes such as DBDS (to probe the anion site) and formycin triphosphate (to probe the ATP site) to confirm the coupling. We will use cellulose and sepharose columns to purify band 3 and to investigate the tightness of its linkage to Na,K-ATPase and will also modify band 3 in red cell membrane vesicles by application of chymotrypsin and trypsin, sulfhydryl reagents and phospholipases. b) Protein-lipid interactions. We have NMR evidence of coupling between the conformation of the Na,K-ATPase and membrane lipids, using the fluorine containing anesthetic, halothane as a lipid probe whose environment can be investigated by 19F NMR. We plan to confirm our preliminary finding and characterize these relationships using this probe as well as the fluorescent lipid probe DPH. c) Water transport studies. We will confirm the evidence of Brown et al that band 3 contains the water tranport channel using 14C-DTNB, and will determine the number of sulfhydryl water inhibition sites per monomer of band 3. We will study the topographical location of the sulfhydryl site by measuring energy transfer between a fluorescent sulfhydryl acceptor and a DBDS donor at the anion site.